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Cyclic nucleotides are important second messengers involved in cellular events, and analogues of this type of molecules are promising drug candidates. Some cyclic nucleotide analogues have become standard tools for the investigation of biochemical and physiological signal transduction pathways, such as the Rp-diastereomers of adenosine and guanosine 3′,5′-cyclic monophosphorothioate, which are competitive inhibitors of cAMP- and cGMP-dependent protein kinases. Next generation analogues exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity, or are caged or photoactivatable for fast and/or targeted cellular imaging. Novel specific nucleotide analogues activating or inhibiting cyclic nucleotide-dependent ion channels, EPAC/GEF proteins, and bacterial target molecules have been developed, opening new avenues for basic and applied research. This review provides an overview of the current state of the field, what can be expected in the future and some practical considerations for the use of cyclic nucleotide analogues in biological systems.
Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.
Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.
Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.
Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
Endothelium-dependent vasodilation is thought to be mediated primarily by the NO/cGMP signaling pathway whereas cAMP-elevating vasodilators are considered to act independent of the endothelial cell layer. However, recent functional data suggest that cAMP-elevating vasodilators such as β-receptor agonists, adenosine or forskolin may also be endothelium-dependent. Here we used functional and biochemical assays to analyze endothelium-dependent, cGMP- and cAMP-mediated signaling in rat aorta. Acetylcholine and sodium nitroprusside (SNP) induced a concentration-dependent relaxation of phenylephrine-precontracted aorta. This response was reflected by the phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a validated substrate of cGMP- and cAMP-dependent protein kinases (cGK, cAK), on Ser157 and Ser239. As expected, the effects of acetylcholine were endothelium-dependent. However, relaxation induced by the β-receptor agonist isoproterenol was also almost completely impaired after endothelial denudation. At the biochemical level, acetylcholine- and isoproterenol-evoked cGK and cAK activation, respectively, as measured by VASP Ser239 and Ser157 phosphorylation, was strongly diminished. Furthermore, the effects of isoproterenol were repressed by eNOS inhibition when endothelium was present. We also observed that the relaxing and biochemical effects of forskolin were at least partially endothelium-dependent. We conclude that cAMP-elevating vasodilators, i.e. isoproterenol and to a lesser extent also forskolin, induce vasodilation and concomitant cyclic nucleotide protein kinase activation in the vessel wall in an endothelium-dependent way.